Altered regulation of lipid and glucose homeostasis, most often in the setting of insulin resistance and obesity, is central to the pathogenesis of common disorders including non-alcoholic fatty liver disease (NAFLD). Because current management options remain limited, the discovery of new metabolic pathways will serve to identify novel opportunities for pharmacologic intervention. This research proposal addresses the unanswered question of whether membrane phospholipids regulate nutrient homeostasis. Our long-term goal is to understand how phospholipid-mediated metabolic control can be leveraged for therapeutic purposes. The objective of this research is to determine the molecular mechanisms whereby sensing of membrane phosphatidylcholine composition by phosphatidylcholine transfer protein (PC-TP) is translated into metabolic control within the liver and oxidative tissues. The central hypothesis is that key regulatory events occur when PC-TP binds specific membrane phosphatidylcholines and then activates thioesterase superfamily member 2 (Them2). The rationale is that the mechanisms of a phosphatidylcholine-sensing pathway should yield new insights into insulin resistance and its complications, including NAFLD. Guided by extensive preliminary data, the central hypothesis will be tested in three specific aims: 1) Demonstrate that Them2 plays a central role in PC-TP-mediated regulation of hepatic lipid and glucose metabolism; 2) Determine the mechanisms by which PC-TP and Them2 limit thermogenesis in brown fat; and 3) Define the influence of phosphatidylcholine molecular species on binding and activation of Them2 by PC-TP. In Aim 1, hyperinsulinemic euglycemic clamp, as well as triglyceride turnover studies in newly created Them2-/- mice will test functions of Them2 downstream of PC-TP. Additional mechanistic insights will be gleaned from experiments in cultured primary mouse hepatocytes, as wel as HEK 293T cells in which expression of endogenous Them2 and PC-TP are silenced using siRNAs. Aim 2 will utilize indirect calorimetry, as well as cultured primary brown adipocytes in order to assess whether PC-TP-Them2 interactions limit mitochondrial fatty acid oxidation. Cultured brown adipocytes wil also be used to evaluate whether PC-TP-Them2 interactions reduce norepinephrine signaling by increasing oxidative stress. In Aim 3, control of PC-TP- Them2 interactions by individual phosphatidylcholine molecular species will be quantified by pulldown assays, surface plasmon resonance and the fatty acyl-CoA thioesterase activity of Them2. The interacting domains of PC-TP and Them2 will be identified by mutational analysis employing a mammalian two-hybrid assay system. Overall, this proposal will elucidate mechanisms of phosphatidylcholine-mediated regulation of lipid and glucose metabolism, which is significant because the fatty acyl composition of the membrane phosphatidylcholines varies in health and disease. These studies are expected to identify new therapeutic targets for the management of for NAFLD, type 2 diabetes and other obesity-associated disorders.